To identify specific protein antigens that functionally contribute to human tumor cell metastasis, our laboratory initiated subtractive immunization approaches to raise unique monoclonal antibodies (mAbs) that inhibit human tumor dissemination. Panels of generated mAbs are screened for their ability to inhibit human tumor dissemination in chick embryo and mouse models of metastasis. Using this approach, we have generated unique antibodies. One of them, mAb 1A5, recognizes a specific member of the tetraspanin family, CD151, and correspondingly inhibits metastasis by > 90%. Another subtractive immunization antibody, mAb 41-2, recognizes a tumor cell transmembrane protein, CDCP1/SIMA-135. This mAb 41-2 inhibits overall metastasis by 60-80%, however the mechanism of mAb 41-2 and that of its target antigen are unknown. Thus, a proposed goal of this renewal is to elucidate the metastasis-inhibition mechanism of mAb 41-2 and to conduct structure- function analysis of its cognate antigen, CDCP1, in order to determine the contributory role of CDCP1 in cancer dissemination. Timed additions of mAb 41-2 into animals followed by quantitative measurements of tumor cell intravascular arrest, apoptosis, survival, extravasation and establishment of secondary foci will determine when, where and how mAb 41-2 blocks malignant function. These approaches will be expanded and confirmed in mouse models of tumor dissemination, including orthotopic implantation approaches. That mAb 41-2's antigen, CDCP1, functions as a survival factor will be tested with mutational analyses of CDCP1's cytoplasmic domain and CDCP1's extracellular CUB domains. The use of combinatorial libraries will be employed to identify unknown natural ligand(s) for CDCP1 and to generate new distinctive antibodies to CDCP1. Another aim of this renewal is to characterize additional pairs of congenic human tumor variants that are similar in tumor-forming ability but differ substantially in their metastasis properties, and to use these cell pairs in new subtractive immunization approaches to generate novel function-blocking antibodies. Panels of mAbs will be screened for differential reactivity and function-blocking properties in our established models. We also propose to initiate potential translational approaches and generate new metastasis-blocking, single chain variable fragment (sc Fv) humanized antibodies that are effective in cancer type-specific orthotopic mouse models and could represent unique reagents for possible targeted tumor therapy.